Designer Zinc Finger Proteins
By Jason Socrates Bardi
For the past several years, Carlos Barbas, who, at 36, holds
the Janet and Keith Kellogg II Chair in Molecular Biology,
has been pursuing one line of research aimed at answering
a simple—but important—question: can one design
proteins to regulate the expression of any human gene?
With zinc fingers, a common structural element found in
proteins, says Barbas, the answer is “yes.”
Barbas has found a set of these small zinc finger protein
"motifs" that each specifically bind to a particular three
base pair sequence of DNA—a codon. By stringing several
of these zinc fingers together, he can create a multiple zinc
finger protein that can bind any sequence of interest, including
unique regulatory regions, and to which he can fuse repressor
and activator proteins to specifically down- and up-regulate
those genes.
Finding Zinc Fingers that Bind
Barbas has accomplished this feat using the same technology
that he developed for his antibody research—phage display.
In his antibody research, which is a separate, thriving
area within his laboratory, Barbas isolates catalytic antibodies—human
immunoglobins that have useful chemical activity in addition
to their antigen specificity.
For instance, Barbas and his colleagues designed the first
commercially available catalytic antibody, 38C2, which can
be made to bind certain markers on a cancer cell and catalyzes
reactions there. This is useful, because patients can be injected
with the antibody and then treated with a highly powerful
anti-cancer agent they otherwise could not tolerate. Many
such agents are too toxic to administer, but they can be rendered
nontoxic by slightly modifying their chemical structure. But
the catalytic antibodies, hinged to surface of the cancerous
cells, can modify them back to their potent form once they
reach the cells.
Barbas and his colleagues find these antibodies through
phage display.
Phage display is a method of generating billions of protein
variants and selecting for those that bind to a particular
target. In the technique, a protein is fused to a viral coat
protien of the phage—a filamentous virus that infects
bacteria. Then the virus is allowed to reproduce in culture,
where it copiously makes new copies of itself. Billions of
variants of a single protein can be studied in this way.
Since the phage virus displays these proteins on the surface
of the virions, it makes them easy to select for in vitro
by simply passing the viral stew over a stationary phase containing
the target substrate. Those that can bind will, and the ones
that bind the best will bind the tightest.
"It became obvious to me that we should use this approach
to evolve proteins that bind specific DNA sequences," says
Barbas. "If we could do that, then we could do almost anything
in the genome that we wanted to—turn genes on or off."
Zinc fingers are a common protein motif in nature because
they bind to DNA. They come in various shapes and sizes, but
they all chelate a zinc ion in their binding domain, and they
all have a long alpha helix that inserts into the major groove
of DNA, making contact with the bases.
Using phage display and oligonucleotide hairpins (short,
single-strand pieces of DNA that twist into tiny helices to
which the zinc fingers can bind), the Barbas laboratory selected
for zinc fingers that bind their target codons with at least
100-fold greater affinity than they bind other sequences that
are a single base change different.
Since three base pairs is the length of a codon and there
are 43=64 possible ways to combine the four types of bases
into a codon, Barbas needed to develop 64 such zinc fingers
to cover all possibilities—a goal which is now in sight.
"We have just a few zinc fingers [left] to find," he says.
Regulating the Regulators
Finding the fingers was only the first step, however. The
next was to combine several of these zinc finger proteins
into a "hand," so that they have a highly selective specificity
for a longer, more unique sequence of DNA. Barbas knew he
had to combine a minimum of six or seven fingers together
in order to recognize 18 to 21 base pairs.
"That DNA address is long enough that it is potentially
found only once in a genome," says Barbas.
Then he had to fuse on the activator or repressor domains
that would do the work of turning the gene on or off. A particular
six zinc finger combination fused with a repressor should
silence the gene’s expression, and the same six zinc
fingers fused to an activator should increase expression.
And he had to design switches that would regulate the activation
or repression of the action of small molecules which, when
bound to the protein, would prevent the zinc fingers from
binding to the DNA. And he had to design reporter systems—green
fluorescent protein and other markers—so that the binding
and to and regulation of the genes could be studied in the
laboratory.
The ability to regulate the regulators is particularly compelling,
because it increases the possibilities for clinical control.
For instance, if one wanted to treat growth hormone deficiency,
one could presumably deliver a transcription factor that would
enhance transcription of growth hormone. But more importantly,
one could couple the transcription factor with a switch that
would activate or inactivate the gene whenever a pill was
swallowed.
"It can be done either way," says Barbas.
Another possibility is to use a tissue-specific switch or
a switch that would be sensitive to endogenous signals. Glucose
levels, for instance, could conceivably be a signal that turns
on or off a gene that encodes for a protein that increases
the production of insulin.
These possibilities are all down the road, but Barbas has
already designed several successful zinc finger transcription
factors to regulate several different genes.
"What we’re doing now," he says, "is applying this
approach to many types of diseases. We have encoded in our
own genes the solutions to many diseases."
"What awaits is just a way to switch on those critical genes."
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